DE3024270C2 - Composition for the determination of human erythropoietin and method for its determination - Google Patents

Description

The invention relates to a composition for the determination of human erythropoietin, consisting of carrier plates and human erythropoietin applied to the surface of these carrier particles, and a method for the determination thereof. The lifespan of human erythrocytes is about 120 days and V ₁₂₀ of total erythrocytes

jo are destroyed daily in the reticulo-endothelial system. At the same time, the same number of erythrocytes is formed in order to keep the number of erythrocytes constant at all times.

The erythrocytes are produced by the maturation and differentiation of the erythroblasts in the bone marrow formed and Erythropoietln Is a factor which by acting on the undlfferenzlerten stem cells initiates their differentiation into erythrocytes. The excretion of erythropoietin is accelerated when a drop in the partial oxygen pressure of body tissues occurs. Thus, Erythropolis is a humoral hormone, which controls the number of erythrocytes made in the bone marrow.

In the case of mammals, all or some of this erythropoietin is supplied by the kidney formed by a glycoprotein containing slallic acid, the molecular weight of which is about 27,000 to 66,000 is appreciated.

The disorders in which human erythrocytes decrease, i. H. Anemia, can be grossly aetiological can be divided into three types. The first kind is the one in which the stem cells, the origin of the Erythrocytes, are abnormal so that the erythrocytes cannot be formed even if the erythropoietin-producing ability is normal; H. splastic anemia. This abnormality of the stem can continue to be divided into such cases as the hypoplastle of the bone marrow, the abnormality of the bone marks themselves, the inadequacy of the places where the erythrocytes are formed, and the decline of the reacti vity for erythropoietin.

In the case of anemic disorders of this type, the body's mechanism that leads to an increase occurs Number of erythrocytes is tending. In effect, so that the amount of erythropoietin becomes extremely or abnormally high. The second type of anemia is that of chronic renal disease. H.

Anemia of the renal disease. Since in this case this is an organic disorder of the kidneys, the producer of the Erythropoietins, erythropoietin cannot be bloated even if the stem cells are normal. Therefore, the number of erythrocytes cannot be increased, resulting in anemia. In In this case there is only a low concentration of erythropolis in the liquid. The third Type of anemia Is the case when both the stem cells and the erythropolytic product volume are soft malfunctions are normal, with the anemic state due to a lack of elsenic, hemorrhage, and vitamin Bu deficiency Autoimmunity-reducing hemolysis and the like. In this case, the amount of the Erythropoietins have a high value and a negative relationship to the amount of hemoglobin.

While the anemia itself can be easily identified by counting the actual number of red blood cells that are Determination of the hemetokrlt value or the determination of hemoglobin can be easily diagnosed can. Is the diagnosis of the cause of the anemic condition, namely whether the cause of the disorder is on one The abnormality of the stem cells is due to an inadequate production of erythropolis Is due to renal disorders or whether the cause is due to reasons other than the above causes, extremely important in the formulation of the treatment regimen and in the Creation of a prognosis for the Krankhell. In each case there is a determination of the erythropoletin in the body fluid is essential for this purpose.

What is known is a biological assessment process that is both complicated, time consuming and costly. In this method, the amount of erythropoietin in the following catfish is calculated. Polythythemic mice or starving rats are injected with Erythropoletin, followed by an injection of Fe

he follows. The amount of erythropolis is calculated from the rate at which the ⁵⁹ Fe is absorbed into the erythrocytes. Although this method has quite excellent specificity, there are disadvantages that a large number of animals must be bred for a long period of time and that an isotope is used. In addition, the method requires considerable labor and expense. It is therefore difficult to apply it to clinical examinations.

To reduce these disadvantages and disruptions, a method has been developed which consists in that erythrocytes are coated with human erythropotetin. Anllerythropolyin antibody is used in This procedure is first neutralized with the erythropoietin contained in the sample, then it is neutralized the sensitive erythrocytes are added and the antigen-body agglutination reaction is inhibited established. However, since this procedure eliminates the need to remove the non-specific hemagglutin, which is contained in human serum, there must be a treatment to absorb the Hemagglutins may be taken and a test to confirm removal performed.

In the reference: G. Müller, Kiln. Biochemie und Laboratoriumsdiagnostik, 1977, pp. 53/54 is a method for the persistence of antibodies or antigens in a body fluid through an agglutination inhibitor indicated, in which instead of erythrocytes serologically inert latex carriers with a particle size from 0.05 to 0.9 μm are coated with antibodies or antigens. Also from DE-OS 22 04 684 is a such a method is known, wherein polymer latex particles of the styrene type having a specific gravity of 1.03 be used. However, there is no suggestion whatsoever of a method for determining human Erythropoiet'n.

The object of the invention is to create a composition for determining the human »- Chen Erythropoietlns, when applied it In contrast to the complicated and tent-robbing so far applied method is possible, both quantitatively and qualitatively with accuracy and exactness that Human erythropoietin contained in a liquid sample by means of simple measures using of a simple device.

This object is achieved according to the invention by creating a composition for Determination of the human erytkiopoietin consisting of carrier plates and on the surface of these Carrier particles applied human erythropoietin, which is characterized in that the carrier particles of synthetic polymer latex particles of the styrene type having a specific gravity of 1.1 to 1.4 consist and from the group of particles of a styrene polymer, styrene copolymer and carboxyl-tert or Amino group-containing carboxyl-tertiary products are selected therefrom. Jö

Furthermore, according to the invention, a method for determining human erythropoietin In human urine or serum nati, the microliter method using a composition, consisting of carrier particles and human applied to the surface of these carrier particles Erythropoietin created, which is characterized by the fact that the carrier particles are made of synthetic polymer latex particles consist of the styrene type with a specific gravity of 1.1 to 1.4 and from the group of Particles of a styrene polymer. Styrene copolymers and containing carboxyl-modified or amino groups carboxyl-enriched products are selected therefrom.

Synthetic polymeric latex particles or particles of a synthetic polymeric latex of the styrene type, which are coated with purified human erythropolis (antigen) can be easily produced. When using these latex particles, the erythropoietin contained in the body fluid can easily and «1 can be easily determined with good accuracy by the passive latex agglutination inhibition reaction, without that complex pretreatments of the test liquid wedge are necessary.

Since the latex coated with erythropoietin agglutinates through reaction with the anti-erythropoietin antibody suffers, agglutination of the antigen-coated latex does not take place when this antibody was previously neutralized with the antigen contained in the sample. If the sample has no antigen contains, the antibody cannot be neutralized. As a result, the agglutination of the with As a result of this antibody, antigen coated latex takes place. This makes it possible to have the presence or To determine the absence of an antigen by means of the passive latcx agglutination inhibition reaction.

When the erythropoietin is determined quantitatively, certain amounts of the antibody are produced on a microsphere to progressively diluted samples in order to neutralize the Lrythropoletin contained in the sample added, then added the antigen-coated latex and the agglutination reaction pattern observed which was formed by the latex and the antibody that remained unneutralized.

This composition only reacts with an anti-erythropolytic antibody which is associated with the test sample was added, and not with other antibodies present in the P-obe or the plasma protein can.

Various synthetic latex particles can be used to make the composition for determining the human erythropoietin can be used according to the invention. Examples of polymers or copolymers, which form such layxes include, for example, polystyrene, carboxylated polystyrene, aminhaltlges carboxylates polystyrene, styrene-butadlene copolymers, carboxylated styrene-butadlene copolymers, styrene-vinylbenzene copolymers and acrylic III-Buladicn-styrene copolymers.

As copolymers of styrene, bcvor / .ugl monomers from the group of chlorostyrene, methyl methacrylate and vinylidene chloride. The synthetic polymurlaiex particles can after a pre-treatment of their surfaces with a non-ionic surface-active. Funds are used. For example, it is preferred to use the particles after having attached thereto an ethylene oxide type nonionic surfactant was absorbed. Examples of suitable non-ionic surface-active agents of the ethylene oxide type are Block copolymers of ethylene oxide and polyoxypropylene glycol. Polyoxyethylene alkyl ethers and polyoxyethylene alkyl aryl ethers.

The synthetic polymeric waxes preferably have an average small diameter knife

from 0.01 to 10 μm, in particular from 0.1 to 1 μm. It is preferred to increase the reproducibility of the results of the determinations. Particles within a relatively narrow range in terms of size distribution, for example a range of 0.2 ± 0.005, to be used. The specific gravity of the latex particles is 1.1 to 1.4 and preferably U1 to 1.3.

5 Although it is possible to use blood as well as urine as the starting material for the extraction of the erythropolis urine is particularly suitable because it represents an excretion and is therefore easily available stands. As indicated in Journal oi Biological Chemistry, Volume 252, pages 5558-5564 (1977), can all known measures, such as ethanol precipitation, acetone precipitation, tannic acid precipitation, lithium oxide precipitation, Salting out with ammonium sulfate, gel filtration, DEAE cellulose chromatography, polyacrylamide gel chromatography and Kaolln adsorption and dissociation for the production of erythropoletin from the the above starting materials can be used.

Since Erythropoieiin can withstand heating to, for example, 100 ⁰ C for 15 minutes. Is it also effective to subject the material to a heat treatment at, for example, about 80 to 100 ^° C. for about 10 to 15 minutes before it is cleaned, so that a large part of the other proteins which may be present are heat-modified. The erythropoletin used in the context of the invention can be one which has been obtained by one of the processes set out above or combinations thereof. However, it is preferred that it be a single protein of high purity because of the potential for side reactions if the purity is low.

The antibody can be obtained by using the purified Erythropoletin obtained in this catfish as an anti-

gene and immunization of animals with the ability to produce antibodies, such as goats, rabbits

and guinea pigs, by inoculating these animals with erythropoietin with a sharp decrease

of their blood are preserved. All animals can be used in this case, provided they are those upon which the antibody lelchi can be obtained.

The composition for the determination of human erylhropoletins according to the invention can be found after a simple process. Example catfish can be obtained. By adding latex particles and the erythropoietin in water, a physiological salt solution or various buffer solutions of pH 5.5 to 10, preferably pH 6.4 to 7.6, at about 4 ° to about 40 ° C. for about 30 minutes to about 24 hours with gentle agitation, preferably with concentrations of the latex particles of 0.05 to 3% and concentrations of the antigen from 50 to 1000 mlu / ml.

The buffer used can, for example, be a phosphate buffered saline solution, such as e.g. B. M / 60 phosphate buffer (pH 7.2) with a content of 0.15 M NaCl (abbreviated to PBS) or glycine-buffered saline. If desired, about 0.01 to about 0.1 ¹ V. of a protein such as ox serum albumin (abbreviated BSA) can preferably be added to the application solution in order to prevent non-specific agglutination. After the coating reaction, the reaction mixture is washed with an aqueous solvent. The antigen not adsorbed on the latex particles can be completely removed by washing the particles with, for example, such buffers. Again, it is recommended that the latex be suspended in a diluent in order to keep the non-antigen coated portion of the latex particles saturated with protein.

The diluent used is a solution obtained by adding about 0.1% BSA to, for example, a glycol-buffered sodium chloride solution or a phosphate-buffered sodium chloride solution. To which further 0.01 to 0.5% sodium hydroxide (NaN ₃ ) was added.

The antigen-coated latex obtained in this catfish can be suspended in a refrigerator • a diluent with a concentration of, for example, about 0.25 wt it can be lyophilized. When carrying out the lyophilization, various amino acids, in particular such amino acids as glycine and sodium glutamai, and / or dextran in such amounts as 0.2 to 2% by weight in the case of the amino acids and 0.3 to 3% by weight in the case of the dextran to the diluent are added as stabilizers, after which the coated latex in liquid nitrogen or liquid Air can be frozen quickly and then iyophllislerl becomes. The storage tent continues to be through the lyophilization is prolonged and the lyophilized anllgenüber / .ogenc latex can usually be stable during about 2 years (four more can be stored.

Measures known per se can be used to carry out the determination. For example ki> nn the level <of human erythropoletins in serum or urine easily after a micro-filtering procedure be determined. A given amount of a diluent of the type set out above Poured in portions onto a microplate and then a given amount of a test sample, such as Serum or urine. Inserted into the first recess of the plate and successively with a thinner diluted. In the same catfish, a dilution series is made using a standard erythropoietic line made to a known concentration. A given amount of the antibody then becomes all Samples and the dilution series added, leaving the samples at room temperature for 15 to 70 minutes to neutralize the antibody. This is followed by the addition and the mixing a given amount of the antigen coated latex. After standing for a given space at room temperature the end point of agglomeration is observed. The absolute amount of erythropoletin can be determined by comparison with the Suindard erythropolis. The invention is explained in more detail below with the aid of examples.

example 1

a) Production of erythropoletin 0.1% phenol were / u de lirin of an aniimlsehcn l'allcnlcn / ugcseUl and after adding the 4lachcn volume

With ethanol, the mixture was left in a refrigerator overnight. Then the was centrifuged at 3000 U / mln for 30 minutes, whereupon the sediment was collected and dissolved in a physiological salt solution and dialyzed again overnight against a physiological salt solution Test tubes were immersed in boiling water for 15 minutes and then centrifuged at high speed. To the supernatant liquid, kaolin washed 5 times in a physiological saline solution was added in an amount of 10%, and after the pH was lowered to 4.8 stirred for 24 hours C the kaolin was separated in the centrifuge at 3000 U / mln, collected and washed with an acetate buffer at pH 4.8 Ils CH ₄ was then suspended in 1 M NLI, weak during ^1; weak mixture at 4.. Stirred hour at room temperature and centrifuged at 3000 U / mln for 30 minutes and then the resulting supernatant liquid was ssigkeit separated. After repeating this operation three times, the supernatant liquids were combined. This sample was again dlalysleri against a solution containing 0.029 M NaII ₂ PO ₄ and 0.029 M NaCl. This sample was then added to a DEAE cellulose which was in _{equilibrium with a solution of 0.029 M NaH 1} PO ₄ and 0.029 M NaCl and was gently stirred at room temperature for 1 hour for its adsorption. Then the DEAE cellulose was collected by centrifugal ion at 2000 U / mln. Washed twice in the above solution and then eluted three times in a solution containing 0.05 M Na ₁ HPO ₄ and 0.15 M NaCl. The elual was then concentrated with 1.yphogcl (polyacrylamide gel from Uelman Instrument Co., USA) and then subjected to gel filtration with Sephadex G-100 (cross-linked dextran from Pharmacia Fine Chemicals, AB. Sweden), and then the active part was collected precipitated again with ethanol, whereupon the precipitate was washed in ethanol and, after dissolution in a physiological salt solution, was redissolved against a physiological salt solution to form the erythropolctin sample.

b) Manufacture of an erythropolis antibody

A sample prepared in the same way as above, except that the heat treatment is not was used as an antigen for immunization. After mixing this up Antigen with Freund's incomplete gel shell mlite was the mixture subcutaneously three times in rabbits with a weight of 2 to 3 kg was injected with an interval of 7 days and then an intramuscular Injection given. Three weeks later, the sample was intravenous in solution in a physiological saline solution injected. Whole blood was drawn from the carotid artery one week after the final injection removed and the serum was separated in the usual catfish and then at 56 ° C for 30 minutes heated. The material was stored at 4 ° C after the addition of 0.1% sodium hydroxide. The received Anti serum formed a band with an antigen by the Ouchterlony method as well as by immunoelectrophoresis. It was thus confirmed that it contained only a single antibody.

A polystyrene latex (SDL 59, specific gravity 1.18. Partial diameter 0.9 microns, product of Takeda Chemical Industry Company) was added to a mixture of 1 volume of a V ₁ , M phosphate buffer (pH 7.2) and 3 volumes of a physiological saline solution ( abbreviated to PBS) so dlsperglert that the small concentration was 0.25 ".. Erythropolyte diluted with PBS to a concentration of 250 mlu / ml (mill! Immunochemical unli / ml) was added to the resulting suspension. The resulting mixture was added at Maintained room temperature for 3 hours and then centrifuged to collect the latex particles, which were washed with PBS and then with a diluent, whereupon the coated particles were suspended in the diluent to a concentration of 0.25 ^ Antierythropoietin antibody agglutinated on a microplate, did not give agglutination when different antibodies from human A. lbumin, human IgG, human transferrin, human haptoglobin, and human / ^ - microglobulin have been added. Furthermore, no agglutination took place when the antigen-coated latex was added after the anti-erythroposiesin antibody had been neutralized by the addition of erythropoietin. Furthermore, after the anti-erythropoietin antibody under identical conditions to a latex which had not been coated with erythropoietin. was added, no agglutination took place. Thus, it was confirmed that this antigen-coated latex specifically reacts with the anti-erythropoletin antibody and agglutinates.

The admonished diluent was obtained by buffering BSA to one with '/ ", M-Phosphai physiological saline solution (pH 7.2) was added to a concentration of 0.1%.

d) lyophilization of the antigen-coated latex

The antigen-coated latex was added to in a diluent with a content of 0.5 wt ^ ¹ M glycine and 0.7 weight s dextran (dextran, molecular weight 200,000 to 300,000, a product of Wako Jyunyaku Co.) a concentration of 2.5 h, suspended. After Rasch-freezing this material in liquid nitrogen, it was lyophilized.

Example 2
Determination of Mrythropolcs

0.025 ml of the diluent was added to each well of the first row of a V-type microplate poured. 0.025 ml of serum was added to the first recess. Furthermore, 75 mlu / ml of standard F. rythropoletin added to the first bore of the second row and progressing with one Diluent diluted according to the Zwcllach-Scrlcnverdünnungsverlahrcn. All cutouts were then with 0.025 ml of an antlerylhropoletin antibody previously diluted with Sistandard Erythropoletin In der Welse mixed, since the sixth recess was the end point. Danr. were the Mixtures kept angry at room temperature for 30 minutes, whereupon 0.05 ml of the obtained in Example 1 c) antigen coated latex was added to each recess. The mixtures were thoroughly j, s mixed in a mill and left to stand at room temperature for at least 10 hours. Of the The point of inhibition of agglutination was then measured and the F. rythropoietin concentration was determined calculated by comparison with the standard Erythropolctln.

The results obtained when the concentration of Erythropoictlns in human serum after this Procedure determined are listed in the following table.

Tabel
Determination of Scrum Erythropoictin (agglutination hcmmwcrl)

No. 1 2 3 4 5 (> 7 R <) I!) Il 12 miu / .nl

Standard solution 75 miu / ml - - - - + + + + + +

Healthy adult - - - - + + + + + + + 37.5

(27 years old, male)

Healthy adult - - - - - - + + + + + + 75

(46 years old, male)

Aplastic anemia _________ - - + 2400

(18 years old. Female)

Aplastic anemia _________- + + 1200

(24 years old, male)

Hypuffficial anemia -______- + + + + 300

(51 years old, female)

Chronic renal insufficiency - + + + + + + + + + + + 2.3

(47 years old, male)

Chronic renal insufficiency --- + + + + + + + + + 9.3

(39 years old, male)

-: unuggled lines +: agfluiinicri

Example 3

A diluent was added to the antigen-coated latex obtained in Example 1d) in such a way that the concentration of Lrtextellchcn was 0.25%, whereupon the operation was carried out as in Example 2 to obtain the contained in the serum To determine erythropolis. The results obtained were the same as in Example 2.
As can be seen from the above results, the blood erythropoietin showed a high value in the case of hypoferritic anemia and an abnormally high value in the case of aplastic anemia, but an abnormally low value in the case of renic anemia. Thus, it can be seen that the determination of erythropolyin in anemic patients is extremely valuable in diagnosing the cause of the anemia in these patients as well as in determining the extent of the disease. The use of the antigen coated latex according to the invention makes it possible to very easily determine the level of erythropoietin within an extremely small amount of the sample and further without the need for any pretreatment of the sample. The method according to the invention is thus particularly suitable for use in clinical diagnosis.

65

Claims (5)

Patent claims: 1. Composition for the determination of human erythropoietin, consisting of carrier particles and human erythropolis applied to the surface of these carrier plates, thereby S indicates that the carrier particles are made of synthetic polymer latex particles of the styrene type with a specific gravity from 1.1 to 1.4 and from the group of particles of a styrene polymer, Styrene copolyraeren and carboxyl-tertiary or amino-containing carboxyl-tertiary products thereof are selected. 2. Composition according to claim 1, characterized in that the latex particles have an average particle diameter of 0.01 to 10 μm. 3. Composition according to claim 1 or 2, characterized in that it consists of a stabilizer Contains amino acids and / or dextran. 4. Composition according to claim 3, characterized in that the stabilizer of glycine in one Concentration of 0.2 to 2% by weight and dextran in a concentration of 0.3 to 3% by weight. 5. Method for the determination of human erythropoietin in human urine or serum according to the milk filter method using a composition consisting of carrier particles and on human erythropolis applied to the surface of these carrier particles, characterized in that that the carrier particles are made of synthetic polymer latex particles of the styrene type with a specific Weight from 1.1 to 1.4 and consist of this group of particles of a styrene polymer, styrene copoly- jo mers and carboxyl-containing or amino-containing carboxyl-containing products selected from these are.